Antibodies to a chemokine expressed in inflamed adenoid

ABSTRACT

The present invention provides nucleotide and amino acid sequences that identify and encode a novel expressed chemokine (ADEC) from inflamed adenoid tissue. The present invention also provides for antisense molecules to the nucleotide sequences which encode ADEC, expression vectors for the production of purified ADEC, antibodies capable of binding specifically to ADEC, hybridization probes or oligonucleotides for the detection of ADEC-encoding nucleotide sequences, genetically engineered host cells for the expression of ADEC, diagnostic tests for inflammation or disease based on ADEC-encoding nucleic acid molecules or antibodies capable of binding specifically to ADEC.

This application is a divisional application of U.S. application Ser.No. 09/203,235, filed Dec. 1, 1998 now U.S. Pat. No. 6,071,701, which isa divisional application of U.S. application Ser. No. 08/862,607, filedMay 23, 1997, now U.S. Pat. No. 5,844,084, which is a divisionalapplication of U.S. application Ser. No. 08/352,324, filed Dec. 7, 1994,now U.S. Pat. No. 5,633,149.

BACKGROUND OF THE INVENTION

Leukocytes including monocytes, macrophages, basophils, and eosinophilsplay important roles in the pathological mechanisms initiated by Tand/or B lymphocytes. Macrophages, in particular, produce powerfuloxidants and proteases which contribute to tissue destruction andsecrete a range of cytokines which recruit and activate otherinflammatory cells.

The investigation of the critical, regulatory processes by which whitecells proceed to their appropriate destination and interact with othercells is underway. The current model of leukocyte movement ortrafficking from the blood to injured or inflamed tissues comprises thefollowing steps. The first step is the rolling adhesion of the leukocytealong the endothelial cells of the blood vessel wall This movement ismediated by transient interactions between selectins and their ligands.A second step involves cell activation which promotes a more stableleukocyte-endothelial cell interaction mediated by the integrins andtheir ligands. This stronger, more stable adhesion precipitates thefinal steps—leukocyte diapedesis and extravasation into the tissues.

The chemokine family of polypeptide cytokines, also known asintercrines, possesses the cellular specificity required to explainleukocyte trafficking in different inflammatory situations First,chemokines mediate the expression of particular adhesion molecules onendotheia cells; and second, they generate gradients of chemoattractantfactors which activate specific cell types. In addition, the chemokinesstimulate the proliferation of specific cell types and regulate theactivation of cells which bear specific receptors. Both of theseactivities demonstrate a high degree of target cell specificity.

The chemokines are small polypeptides, generally about 70-100 aminoacids (aa) in length, 8-11 kD in molecular weight and active over a1-100 ng/ml concentration range. Initially, they were isolated andpurified from inflamed tissues and characterized relative to theirbioactivity. More recently, chemokines have been discovered throughmolecular cloning techniques and characterized by structural as well asfunctional analysis.

The chemokines are related through a four cysteine motif which is basedprimarily on the spacing of the first two cysteine residues in themature molecule. Currently the chemokines are assigned to one of twofamilies, the C-X-C chemokines (α) and the C-C chemokines (β). Althoughexceptions exist, the C-X-C chemokines generally activate neutrophilsand fibroblasts while the C-C chemokines act on a more diverse group oftarget cells which include monocytes/macrophages, basophils,eosinophils, T lymphocytes and others. The known chemokines of bothfamilies are synthesized by many diverse cell types as reviewed inThomson A. (1994) The Cytokine Handbook, 2d Ed Academic Press, NY. Thetwo groups of chemokines will be described in turn.

The archetypal and most extensively studied C-X-C chemokine is plateletfactor 4 (PF4). This 70 aa protein displays the definitive fourcysteines and is released along with platelet derived growth factor(PDGF), transforming growth factor β (TGF-β) and β-thromboglobulin(β-TG) from the granules of stimulated platelets. This homotetramericmolecule shares structural similarity with interleukin-8 (IL-8), inducesthe migration of fibroblasts, neutrophils and monocytes, and bindsheparin. PF4 provides the biological model for a link among thrombosis,inflammation, and wound healing.

Other chemokines found in the platelet a granule include β-TG,connective tissue activating protein III (CTAP-III) and neutrophilactivating peptide 2 (NAP-2). All three peptides are derived from thedifferential processing of a precursor molecule, platelet basic protein(PBP). β-TG is an 81 aa, highly basic protein which influences themigration of fibroblasts but has no effect on neutrophils or monocytes.CATP-III is 85 aa long, and aa 4-85 are identical to β-TG. SinceCATP-III is the primary protein in the a granule and its role as apurified protein has not been elucidated, it may be a secondaryprecursor, inactive until further processed NAP-2 appears to attractneutrophils but not monocytes.

Nonplatelet C-X-C chemokines include IL-8, γ interferon inducibleprotein (IP-10), melanocyte growth stimulatory activity (MGSA or gro)proteins, epithelial derived neutrophil attractant-78 (ENA-78),granulocyte chemotactic protein-2 GPC-2) and stromal cellderivedfactors-1α and 1β (SDF-1α and -1β). IL-8 (also known as NAP-1) issecreted by monocytes/macrophages, neutrophils, fibroblasts, endothelialcells, keratinocytes and T lymphocytes in response to proinflammatorycytokines, IL-1 and 3, IFN-γ and TNF, as well as endotoxins, mitogens,particulates, bacteria and viruses. IL-8 stimulates acute inflammationincluding the upregulation of both neutrophil adhesion and keratinocytegrowth and the downregulation of histamine production by basophils.

IP-10 is a 10 kD protein of undefined function whose mRNA has been foundin monocytes, fibroblasts and epdothelial cells. Monocytes,keratinocytes and activated T cells secrete IP-10 protein which has beenlocalized to sites of delayed hypersensitivity reactions. The cDNA ofMGSA/gro a produces a 15 kD protein which appears in fibroblasts. Itstranscription is growth related, and it functions as an autocrine growthfactor. The distinct and non-allelic forms, gro β and gro γ, are 90% and86% identical to gro α, respectively. Recombinant gro α proteins attractand activate neutrophils. ENA-78 was purified from supernatants of lungalveolar cells. Like gro α, it attracts and activates neutrophils invitro.

GCP-2 is a 6 kD protein isolated from the supernatants of osteosarcomacells. GCP-2 exists in various N-terminally truncated forms, and itattracts and activates neutrophils in vitro and causes granulocyteaccumulation in vivo. SDF-1α and -1β are newly isolated cDNAs whichencode secreted molecules and type I membrane proteins.

Current techniques for diagnosis of abnormalities in inflamed ordiseased tissues mainly rely on observation of clinical symptoms orserological analyses of body tissues or fluids for hormones,polypeptides or various metabolites. Patients often manifest no clinicalsymptoms at early stages of disease or tumor development Furthermore,serological analyses do not always differentiate between invasivediseases and genetic syndromes which have overlapping or very similarranges. Thus, development of new diagnostic techniques comprising smallmolecules such as the expressed chemokines are important to provide forearly and accurate diagnoses, to give a better understanding ofmolecular pathogenesis, and to use in the development of effectivetherapies.

The chemokine molecules were reviewed in Schall T J (1994) ChemotacticCytokines: Targets for Therapeutic Development. International BusinessCommunications, Southborough, Mass., pp 180-270; and in Paul W E (1993)Fundamental Immunology, 3rd Ed. Raven Press, NYC, pp 822-826.

SUMMARY OF THE INVENTION

The subject invention provides a nucleotide sequence which uniquelyencodes a novel human protein from inflamed adenoid The new gene, whichis known as adenoid expressed chemokine, or adec (Incyte Clone No.20293), encodes a polypeptide designated ADEC, of the C-X-C chemokinefamily. The invention also comprises diagnostic tests for inflammatoryconditions which include the steps of testing a sample or an extractthereof with adec DNA, fragments or oligomers thereof. Aspects of theinvention include the antisense DNAs of adec, cloning or expressionvectors containing adec; host cells or organisms transformed withexpression vectors containing adec; a method for the production andrecovery of purified ADEC from host cells; and purified ADEC.

DESCRIPTION OF THE FIGURES

FIG. 1 displays the nucleotide sequence for adec and the predicted aminoacid (aa) sequence of the adenoid expressed chemokine, ADEC.

FIG. 2 shows the aa alignment of ADEC with other human chemokines of theC-X-C family. Alignments shown were produced using the multisequencealignment program of DNASTAR software (DNASTAR Inc, Madison Wis.).

FIG. 3 shows a relatedness tree of human C-X-C chemokines. Thephylogenetic tree was generated by phylogenetic tree program of DNASTARsoftware (DNASTAR Inc, Madison Wis.) using the Clustal method with thePAM250 residue weight table.

FIG. 4 displays an analysis of hydrophobicity and immunogeniccharacteristics of ADEC based on the predicted aa sequence andcomposition.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “adenoid expressed chemokine” or ADEC, refers to apolypeptide, a naturally occurring ADEC or active fragments thereof,which is encoded by an mRNA transcribed from ADEC cDNA of a particularSeq ID No.

“Active” refers to those forms of ADEC which retain the biologic and/orimmunologic activities of naturally occurring ADEC.

“Naturally occurring ADEC” refers to ADEC produced by human cells thathave not been genetically engineered and specifically contemplatesvarious ADEC forms arising from post-translational modifications of thepolypeptide including but not limited to acetylation, carboxylation,glycosylation, phosphorylation, lipidation and atylation.

“Derivative” refers to polypeptides derived from naturally occurringADEC by chemical modifications such as ubiquitination, labeling (e.g.,with radionuclides, various enzymatic modifications), pegylation(derivatization with polyethylene glycol) or by insertion orsubstitution by chemical synthesis of aa such as omithine, which do notnormally occur in human proteins.

“Recombinant variant” refers to any polypeptide differing from naturallyoccurring ADEC by aa insertions, deletions, and substitutions, createdusing recombinant DNA techniques Guidance in determining which aaresidues may be replaced, added or deleted without abolishing activitiesof interest, cell adhesion and chemotaxis, may be found by comparing thesequence of the particular ADEC with that of homologous cytokines andminimizing the number of aa sequence changes made in regions of highhomology.

Preferably, aa substitutions are the result of replacing one aa withanother aa having similar structural and/or chemical properties, such asthe replacement of a leucine with an isoleucine or valine, an aspartatewith a glutamate, or a threonine with a serine, i.e., conservative aareplacements. Insertions or deletions are typically in the range ofabout 1 to 5 aa. The variation allowed may be experimentally determinedby systematically making insertions, deletions, or substitutions of aain ADEC using recombinant DNA techniques and assaying the resultingrecombinant variants for activity.

Where desired, a “signal or leader sequence” can direct the polypeptidethrough the membrane of a cell. Such a sequence may be naturally presenton the polypeptides of the present invention or provided fromheterologous protein sources by recombinant DNA techniques.

A polypeptide “fragment,” “portion,” or “segment” is a stretch of aaresidues of at least about 5 amino acids, often at least about 7 aa,typically at least about 9 to 13 aa, and, in various embodiments, atleast about 17 or more am To be active, ADEC polpeptide must havesufficient length to display biologic and/or immunologic activity.

An “oligonucleotide” or polynucleotide “fragment”, “portion,” or“segment” is a stretch of nucleotide residues which is long enough touse in polymerase chain reaction (PCR) or various hybridizationprocedures to amplify or simply reveal related parts of mRNA or DNAmolecules.

The present invention includes purified ADEC polypeptides from naturalor recombinant sources, cells transformed with recombinant nucleic acidmolecules encoding ADEC. Various methods for the isolation of the ADECpolypeptides may be accomplished by procedures well known in the art.For example, such polypeptides may be purified by immunoaffinitychromatography by employing the antibodies provided by the presentinvention. Various other methods of protein purification well known inthe art include those described in Deutscher M (1990) Methods inEnzymology Vol. 182, Academic Press, San Diego; and Scopes R (1982)Protein Purification: Principles and Practice. Springer-Verlag, NYC,both incorporated herein by reference.

“Recombinant ” may also refer to a polynucleotide which encodes ADEC andis prepared using recombinant DNA techniques. The DNA which encodes ADECmay also include allelic or recombinant variants and mutants thereof.

“Oligonucleotides” or “nucleic acid probes” are prepared based on thecDNA sequence which encodes ADEC provided by the present invention.Oligonucleotides comprise portions of the DNA sequence having at leastabout 15 nucleotides, usually at least about 20 nucleotides. Nucleicacid probes comprise portions of the sequence having fewer nucleotidesthan about 6 kb, usually fewer than about 1 kb. After appropriatetesting to eliminate false positives, these probes may be used todetermine whether mRNA encoding ADEC is present in a cell or tissue orto isolate,similar nucleic acid sequences from chromosomal DNA asdescribed by Walsh P S et al (1992) PCR Methods Appl 1:241-250.

Probe may be derived from naturally occurring or recombinant single- ordouble-stranded nucleic acids or be chemically synthesized. They may belabeled by nick translation, Klenow fill-in reaction, PCR or othermethods well known in the art. Probes of the present invention, theirpreparation and/or labeling are elaborated in Sambrook J et al (1989)Molecular Cloning: A Laboratory Manual, 2d Ed, Cold Spring Harbor; orAusubel F M et al (1989) Current Protocols in Molecular Biology, Vol 2,John Wiley & Sons, both incorporated herein by reference.

Alternatively, recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code.

Various codon substitutions, such as the silent changes which producevarious restriction sites, may be introduced to optimize cloning into aplasmid or viral vector or expression in a particular prokaryotic oreukaryotic system. Mutations may also be introduced to modify theproperties of the polypeptide, to change ligand-binding affinities,interchain affinities, or polypeptide degradation or turnover rate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a nucleotide sequence uniquelyidentifying a novel chemokine of the C-X-C family, ADEC, which is highlyexpressed in inflamed adenoid. Because ADEC is specifically expressed inthe tissue from which it was identified and has not been found in othertissues, the nucleic acid (adec), polypeptide (ADEC) and antibodies toADEC are useful in diagnostic tests for inflamed or diseased adenoidExcessive expression of ADEC leads to activation of neutrophils andfibroblasts which respond by producing abundant proteases and othermolecules which can lead to tissue damage or destruction. Therefore, adiagnostic test for excess expression of ADEC can accelerate diagnosisand proper treatment of inflammation before extensive tissue damage ordestruction occurs.

The nucleotide sequences encoding ADEC (or their complement) havenumerous applications in techniques known to those skilled in the art ofmolecular biology. These techniques include use as hybridization probes,use as oligomers for PCR, use for chromosome and gene mapping, use inthe recombinant production of ADEC, and use in generation of anti-senseDNA or RNA, their chemical analogs and the like. Uses of nucleotidesencoding ADEC disclosed herein are exemplary of known techniques and arenot intended to limit their use in any technique known to a person ofordinary skill in the art. Furthermore, the nucleotide sequencesdisclosed herein may be used in molecular biology techniques that havenot yet been developed, provided the new techniques rely on propertiesof nucleotide sequences that are currently known, e.g., the tripletgenetic code, specific base pair interactions, etc.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of ADEC-encodingnucleotide sequences, some bearing minimal nucleotide sequence homologyto the nucleotide sequence of any known and naturally occurring gene maybe produced. The invention has specifically contemplated each and everypossible variation of nucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the nucleotide sequence of naturally occurring ADEC,and all such variations are to be considered as being specificallydisclosed.

Although nucleotide sequences which encode ADEC and/or ADEC variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring ADEC gene under stringent conditions, it may beadvantageous to produce nucleotide sequences encoding ADEC or ADECderivatives possessing a substantially different codon usage. Codons canbe selected to increase the rate at which expression of the polypeptidein a particular prokaryotic or eukaryotic expression host in accordancewith the frequency with which particular codons are utilized by thehost. Other reasons for substantially altering the nucleotide sequenceencoding ADEC and/or ADEC derivatives without altering the encoded aasequence include the production of RNA transcripts having more desirableproperties, e.g., a greater half-life, than transcripts produced fromthe naturally occurring nucleotide sequence.

Nucleotide sequences encoding ADEC may be joined to a variety of othernucleotide sequences by means of well established recombinant DNAtechniques (cf Sambrook J et al. (1989) Molecular Cloning: A LaboratoryManual, 2d Ed, Cold Spring Harbor).

Useful nudeotide sequences for joining to adec include an assortment ofcloning vectors, e.g., plasmids, cosmids, lambda phage derivatives,phagemids, and the like, that are known in the art. Vectors of interestinclude expression vectors, replication vectors, probe generationvectors, sequencing vectors, and the like. In general, vectors ofinterest may contain an origin of replication functional in at least oneorganism, convenient restriction endonucleasm sensitive sites, andselectable markers for the host cell.

Another aspect of the subject invention is to provide for adec-specificnucleic acid hybridization probes capable of hybridizing with naturallyoccurring nucleotide sequences encoding ADEC. Such probes for thedetection of similar chemokine encoding sequences should preferablycontain at least 50% of the nucleotides from a C-X-C encoding sequence.The hybridization probes of the subject invention may be derived fromthe nucleotide sequences of the SEQ ID NO 1 or from genomic sequencesincluding promoters, enhancer elements and introns of naturallyoccurring adec. Hybridization probes may be labeled by a variety ofreporter groups, including radionuclides such as ³²P or ³⁵S, orenzymatic labels such as alkaline phosphatase, coupled to the probe viaavidin/biotin coupling systems, and the like.

PCR as described in U.S. Pat. Nos. 4,965,188 and 4,683,195 and 4,800,195provides additional uses for oligonucleotides based upon the nucleotidesequences which encode ADEC. Such probes used in PCR may be ofrecombinant origin, may be chemically synthesized, or may be a mixtureof both and comprise a discrete nucleotide sequence for diagnostic useor a degenerate pool of possible sequences for identification of closelyrelated genomic sequences.

Other means of producing adec-specific hybridization probes include thecloning of nucleic acid sequences encoding ADECs and ADEC derivativesinto vectors for the production of mRNA probes. Such vectors are knownin the art and are commercially available and may be used to synthesizeRNA probes in vitro by means of the addition of the appropriate RNApolymerase as T7 or SP6 RNA polymerase and the appropriate radioactivelylabeled nucleotides.

It is now possible to produce a DNA sequence, or portions thereof,encoding ADEC and ADEC derivatives entirely by synthetic chemistry,after which the gene can be inserted into any of the many available DNAvectors using reagents, vectors and cells that are known in the art atthe time of the filing of this application. Moreover, syntheticchemistry may be used to introduce mutations into the adec sequence orany portion thereof.

The nucleotide sequence can be used to construct an assay to detectinflammation and disease associated with abnormal levels of expressionof ADEC. The nucleotide sequence can be labeled by methods known in theart and added to a fluid or tissue sample from a patient underhybridizing conditions. After an incubation period, the sample is washedwith a compatible fluid which optionally contains a dye (or other labelrequiring a developer) if the nucleotide has been labeled with anenzyme. After the compatible fluid is rinsed off, the dye is quantitatedand compared with a standard If the amount of dye is significantlyelevated, the nucleotide sequence has hybridized with the sample. Ifadec is present at an abnormal level, the assay indicates the presenceof inflammation and/or disease.

The nudeotide sequence for adec can be used to construct hybridizationprobes for mapping that gene. The nucleotide sequence provided hereinmay be mapped to a chromosome and specific regions of a chromosome usingwell known genetic and/or chromosomal mapping techniques. Thesetechniques include in situ hybridization, linkage analysis against knownchromosomal markers, hybridization screening with libraries orflow-sorted chromosomal preparations specific to known chromosomes, andthe like. The technique of fluorescent in situ hybridization ofchromosome spreads has been described, among other places, in Verma etal (1988) Human Chromosomes: A Manual of Basic Techniques, PergamonPress, NYC.

Fluorescent in situ hybridization of chromosomal preparations and otherphysical chromosome mapping techniques may be correlated with additionalgenetic map data. Examples of genetic map data can be found in O'Brien(1990) Genetic Maps: Locus Maps of Complex Genomes, Book 5: Human Maps,Cold Spring Harbor Laboratory Press. Correlation between the location ofadec on a physical chromosomal map and a specific disease (orpredisposition to a specific disease) can help delimit the region of DNAassociated with that genetic disease. The nucleotide sequence of thesubject invention may be used to detect differences in gene sequencebetween normal, carrier and affected individuals.

Nucleotide sequences encoding ADEC may be used to produce purified ADECusing well known methods of recombinant DNA technology. Among the manypublications that teach methods for the expression of genes after theyhave been isolated is Goeddel (1990) Gene Expression Technology, Methodsand Enzymology. Vol 185, Academic Press, San Diego. ADEC may beexpressed in a variety of host cells, either prokaryotic or eukaryoticHost cells may be from species either the same or different from thespecies in which adec nudeotide sequences are endogenous Advantages ofproducing ADEC by recombinant DNA technology include obtaining highlyenriched sources of the proteins for purification and the availabilityof simplified purification procedures.

Cells transformed with DNA encoding ADEC may be cultured underconditions suitable for the expression of the ADEC and the recovery ofthe protein from the cell culture. ADEC produced by a recombinant cellmay be secreted or may be contained intracellulariy, depending on theparticular genetic construction used. In general, it is more convenientto prepare recombinant proteins in secreted form. Purification stepsdepend on the nature of the production process used and the particularADEC produced.

In addition to recombinant production, ADEC fragments may be produced bydirect peptide synthesis using solid-phase techniques (cf Stewart et al(1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco;Merrifield J (1963) J Am Chem Soc 85:2149-2154).

In vitro protein synthesis may be performed using manual techniques orby automation Automated synthesis may be achieved, for example, usingApplied Biosystems 431 A Peptide Synthesizer (Foster City, Calif.) inaccordance with the instructions provided by the manufacturer. Variousfragments of ADEC may be chemically synthesized separately and combinedusing chemical methods to produce the full length molecule.

ADEC for antibody induction does not need to have biological activity;however, it must be immunogenic. Peptides used to induce ADEC specificantibodies may have an aa sequence consisting of at least five aa,preferably at least 10 aa. They should mimic a portion of the aasequence of ADEC and may contain the entire aa sequence of the naturallyoccurring molecule. Short stretches of ADEC aa may be fused with thoseof another protein such as keyhole limpet hemocyanin and the chimericmolecule used for antibody production.

Antibodies specific for ADEC may be produced by inoculation of anappropriate animal with the polypeptide or an antigenic fragment Anantibody is specific for ADEC if it is produced against an epitope ofthe polypeptide and binds to at least part of the natural or recombinantprotein. Induction of antibodies includes not only the stimulation of animmune response by injection into animals, but also analogous steps inthe production of synthetic antibodies or other specific-bindingmolecules such as the screening of recombinant immunoglobulin libraries(cf Orlandi R et al (1989) PNAS 86:3833-3837, or Huse W D et al (1989).Science 256:1275-1281) or the in vitro stimulation of lymphocytepopulations. Current technology (Winter G, Milstein C (1991) Nature349:293-299) provides for a number of highly specific binding reagentsbased on the principles of antibody formation. These techniques may beadapted to produce molecules specifically binding ADEC.

An additional embodiment of the subject invention is the use ofADEC-specific antibodies, inhibitors, receptors or their analogs asbioactive agents to treat inflammation or disease of the adenoidincluding, but not limited to, tonsilitis, Epstein-Barr virus, Hodgkin'sdisease, various neoplasms or nonspecific pharyngitis. Compositionscomprising the above mentioned molecules may be administered in asuitable therapeutic dose determined by any of several methodologiesincluding clinical studies on mammalian species to determine maximaltolerable dose and on normal human subjects to determine safe dose.Additionally the bioactive agent may be complexed with a variety of wellestablished compounds or compositions which enhance stability orpharmacological properties such as half-life. It is contemplated thatthe therapeutic, bioactive agent may be delivered orally via lozenges,syrups, sprays or topical application, by subcutaneous injection,airgun, etc.

The examples below are provided to illustrate the subject invention.These examples are provided by way of illustration and are not includedfor the purpose of limiting the invention.

EXAMPLES I Isolation of mRNA and Construction of cDNA Libraries

The adec cDNA sequence was identified among the sequences comprising theinflamed adenoid library. This library was constructed from mixedadenoid and tonsil lymphoid tissue surgically removed from a childduring a tonsilectomy. The adenoid tissue was obtained from Universityof California at Los Angeles and frozen for future use. The frozentissue was ground in a mortar and pestle and lysed immediately in buffercontaining guanidinium isothiocyanate (cf Chirgwin J M et al (1979)Biochemistry 18:5294). Lysis was followed by several phenol-chloroformextractions and ethanol precipitations. Poly-A+ mRNA was isolated usingbiotinylated oligo d(T) and streptavidin coupled to paramagneticparticles (Promega, Poly(A) Tract Isolation System).

The poly A mRNA from the inflamed adenoid tissue was used by StratageneInc. (11011 N. Torrey Pines Rd., La Jolla, Calif. 92037) to construct acDNA library. cDNA synthesis was primed using oligo dT and/or randomhexamers. Synthetic adapter oligonucleotides were ligated onto cDNA endsenabling its insertion into the UNI-ZAP™ vector system (StratageneInc.). This allows high efficiency unidirectional (sense orientation)lambda library construction and the convenience of a plasmid system withblue/white color selection to detect clones with cDNA insertions.

The quality of the each cDNA library was screened using either DNAprobes or antibody probes, and then the PBLUESCRIPT® phagemid(Stratagene Inc.) was rapidly excised in living cells. The phagemidallows the use of a plasmid system for easy insert characterization,sequencing, site-directed mutagenesis, the creation of unidirectionaldeletions and expression of fusion proteins. Phage particles from eachlibrary were infected into the E.coli host strain XL-1 BLUE® (StratageneInc.). The high transformation efficiency of XL1-BLUE increases theprobability of obtaining rare, under-represented clones from the cDNAlibrary.

II Isolation of cDNA Clones

The phagemid forms of individual cDNA clones were obtained by the invivo excision process, in which XL1 -BLUE was coinfected with an f1helper phage. Proteins derived from both lambda phage and f1 helperphage initiated new DNA synthesis from defined sequences on the lambdatarget DNA and created a smaller, single stranded circular phagemid DNAmolecule that included all DNA sequences of the PBLUESCRIPT plasmid andthe cDNA insert. The phagemid DNA was released from the cells andpurified, then used to re-infect fresh bacterial host cells (SOLR,Stratagene Inc.), where the double stranded phagemid DNA was produceBecause the phagemid carries the gene for β-lactamase, the newlytransformed bacteria were selected on medium containing ampicillin.

Phagemid DNA was purified using the QIAWELL-8 plasmid purificationsystem from QIAGEN DNA purification system (QIAGEN Inc., 9259 Eton Ave.,Chatsworth, Calif. 91311). This technique provides a rapid and reliablehigh-throughput method for lysing the bacterial cells and isolatinghighly purified phagemid DNA. The DNA eluted from the purification resinwas suitable for DNA sequencing and other analytical manipulations.

III Sequencing of cDNA Clones

The cDNA inserts from random isolates of the inflamed adenoid librarywere sequenced in part. Methods for DNA sequencing are well known in theart. Conventional enzymatic methods employed DNA polymerase Klenowfragment, SEQUENASE DNA polymerase (US Biochemical Corp, Cleveland,Ohio) or Taq polymerase to extend DNA chains from an oligonucleotideprimer annealed to the DNA template of interest. Methods have beendeveloped for the use of both single- and double- stranded templates.The chain termination reaction products were electrophoresed onurea-acrylamide gels and detected either by autoradiography (forradionuclide-labeled precursors) or by fluorescence (forfluorescent-labeled precursors). Recent improvements in mechanizedreaction preparation, sequencing and analysis using the fluorescentdetection method have permitted expansion in the number of sequencesthat can be determined per day (using machines such as the CATALYST 800DNA sequencer and the Applied Biosystems 373 DNA sequencer).

IV Homology Searching of cDNA Clones and Deduced Protein

Each sequence so obtained was compared to sequences in GenBank using asearch algorithm developed by Applied Biosystems Inc. and incorporatedinto the INHERIT™ 670 sequence analysis system. In this algorithm,Pattern Specification Language (developed by TRW Inc.) was used todetermine regions of homology. The three parameters that determine howthe sequence comparisons run were window size, window offset, and errortolerance. Using a combination of these three parameters, the DNAdatabase was searched for sequences containing regions of homology tothe query sequence, and the appropriate sequences were scored with aninitial value. Subsequently, these homologous regions were examinedusing dot matrix homology plots to distinguish regions of homology fromchance matches. Smith-Waterman alignments were used to display theresults of the homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT 670 sequence analysis system in a way similar to that used inDNA sequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches.

The nucleotide and amino acid sequences for the adenoid expressedchemokine, ADEC are shown in FIG. 1.

V Identification and Full Length Sequencing of the Gene

From all of the randomly picked and sequenced clones of the inflamedadenoid library, adec sequences were homologous to)but clearly differentfrom any known C-X-C chemokine molecule. The nucleotide sequence foradec was found within Incyte clone 20293. When all three possiblepredicted translations of the sequence were searched against proteindatabases such as SwissProt and PIR, no exact matches were found to thepossible translations of adec. FIG. 2 shows the comparison of ADEC withother a chemokine molecules; substantial regions of homology includingthe C-X-C motif are shaded. The phylogenetic analysis, however, (FIG. 3)shows that adec is not very closely related to other well characterizedhuman C-X-C chemokines. The most related of these molecules clustertogether at the right hand side of the figure. It appears that adec mayrepresent a new subfamily or variant of the C-X-C chemokines.

VI Antisense Analysis

Knowledge of the correct, complete cDNA sequences of the novel expressedchemokine genes will enable their use in antisense technology in theinvestigation of gene function. Either oligonucleotides, genomic or cDNAfragments comprising the antisense strand of adec can be used either invitro or in vivo to inhibit expression of the specific protein. Suchtechnology is now well known in the art, and probes can be designed atvarious locations along the nucleotide sequence. By treatment of cellsor whole test animals with such antisense sequences, the gene ofinterest can be effectively turned off. Frequently, the function of thegene can be ascertained by observing behavior at the cellular, tissue ororganismal level (e.g. lethality, loss of differentiated function,changes in morphology, etc.).

In addition to using sequences constructed to interrupt transcription ofthe opening reading frame, modifications of gene expression can beobtained by designing antisense sequences to intron regions,promoter/enhancer elements, or even to trans-acting regulatory genes.Similarly, inhibition can be achieved using Hogeboom base-pairingmethodology, also known as “triple helix” base pairing.

VII Expression of ADEC

Expression of adec may be accomplished by subcloning the cDNA into anappropriate expression vector and transfecting this vector into anappropriate expression host. In this particular case, the cloning vectorpreviously used for the generation of the tissue library also providesfor direct expression of the included sequence in E coli. Upstream ofthe cloning site, this vector contains a promoter for β-galactosidase,followed by sequence containing the amino-terminal Met and thesubsequent 7 residues of β-galactosidase. Immediately following theseeight residues is an engineered bacteriophage promoter useful forartificial priming and transcription and a number of unique restrictionsites, including Eco RI, for cloning.

Induction of the isolated bacterial strain with IPTG using standardmethods will produce a fusion protein corresponding to the first sevenresidues of β-galactosidase, about 15 residues of “linker”, and thepeptide encoded within the cDNA. Since cDNA clone inserts are generatedby an essentially random process, there is one chance in three that theincluded cDNA will lie in the correct frame for proper translation. Ifthe cDNA is not in the proper reading frame, it can be obtained bydeletion or insertion of the appropriate number of bases by well knownmethods including in vitro mutagenesis, digestion with exonuclease IIIor mung bean nuclease, or oligonucleotide linker inclusion.

Adec cDNA can be shuttled into other vectors known to be useful forexpression of protein in specific hosts. Oligonucleotide amplimerscontaining cloning sites as well as a segment of DNA sufficient tohybridize to stretches at both ends of the target cDNA (25 bases) can besynthesized chemically by standard methods. These primers can then beused to amplify the desired gene segments by PCR. The resulting new genesegments can be digested with appropriate restriction enzymes understandard conditions and isolated by gel electrophoresis. Alternately,similar gene segments can be produced by digestion of the cDNA withappropriate restriction enzymes and filling in the missing gene segmentswith chemically synthesized oligonucleotides. Segments of the codingsequence from more than one gene can be ligated together and cloned inappropriate vectors to optimize expression of recombinant sequence.

Suitable expression hosts for such chimeric molecules include but arenot limited to mammalian cells such as Chinese Hamster Ovary and human293 cells, insect cells such as Sf9 cells, yeast cells such asSaccharomyces cerevisiae, and bacteria such as E. coli. For each ofthese cell systems, a useful expression vector may also include anorigin of replication to allow propagation in bacteria and a selectablemarker such as the β-lactamase antibiotic resistance gene to allowselection in bacteria. In addition, the vectors may include a secondselectable marker such as the neomycin phosphotransferase gene to allowselection in transfected eukaryotic host cells. Vectors for use ineukaryotic expression hosts may require RNA processing elements such as3′ polyadenylation sequences if such are not part of the cDNA ofinterest.

Additionally, the vector may contain promoters or enhancers whichincrease gene expression. Such promoters are host specific and includeMMTV, SV40, or metallothionine promoters for CHO cells, trp, lac, tac orT7 promoters for bacterial hosts; or alpha factor, alcohol oxidase orPGH prorrtoters for yeast. Transcription enhancers, such as the RSVenhancer, may be used in mammalian host cells. Once homogeneous culturesof recombinant cells are obtained through standard culture methods,large quantities of recombinantly produced ADEC can be recovered fromthe conditioned medium and analyzed using chromatographic methods knownin the art.

VIII Isolation of Recombinant ADEC

ADEC may be expressed as a chimeric protein with one or more additionalpolypeptide domains added to facilitate protein purification. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAG extension/affinity purification system (Immunex Corp., SeattleWash.). The inclusion of a cleavable linker sequence (such as Factor XAor enterokinase) between the purification domain and the ADEC-encodingsequence may be useful to facilitate production of ADEC.

IX Production of ADEC-Specific Antibodies

Two approaches are utilized to raise antibodies to ADEC, and eachapproach is useful for generating either polyclonal or monoclonalantibodies. In one approach, denatured ADEC from the reverse phase HPLCseparation is obtained in quantities up to 75 mg. This denatured proteincan be used to immunize mice or rabbits using standard protocols; about100 micrograms are adequate for immunization of a mouse, while up to 1mg might be used to immunize rabbit For identifying mouse hybridomas,the denatured protein can be radioiodinated and used to screen potentialmurine β-cell hybridomas for those which produce antibody. Thisprocedure requires only small quantities of protein, such that 20 mgwould be sufficient for labeling and screening of several thousandclones.

In the second approach, the amino acid sequence of ADEC, as deduced fromtranslation of the cDNA, is analyzed to determine regions of highimmunogenicity. Oligopeptides comprising hydrophilic regions, as shownin FIG. 4, are synthesized and used in suitable immunization protocolsto raise antibodies. Analysis to select appropriate epitopes isdescribed by Ausubel F M et al (1989, Current Protocols in MolecularBiology, Vol 2, John Wiley & Sons). The optimal amino acid sequences forimmunization are usually at the C-terminus, the N-terminus and thoseintervening, hydrophilic regions of the polypeptide which are likely tobe exposed to the external environment when the protein is in itsnatural conformation.

Typically, selected peptides, about 15 residues in length, aresynthesized using an Applied Biosystems Peptide Synthesizer Model 431 Ausing fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH,Sigma) by reaction with M- maleimidobenzoyl-N-hydroxysuccinimide ester(MBS; cf. Ausubel FM et al, supra). If necessary, a cysteine may beintroduced at the N-terminus of the peptide to permit coupling to KLH.Rabbits are immunized with the peptide-KLH complex in complete Freund'sadjuvant. The resulting antisera are tested for antipeptide activity bybinding the peptide to plastic, blocking with 1% BSA, reacting withantisera, washing and reacting with labeled (radioactive orfluorescent), affinity purified, specific goat anti-rabbit IgG.

Hybridomas may also be prepared and screened using standard techniques.Hybridomas of interest are detected by screening with labeled ADEC toidentify those fusions producing the monoclonal antibody with thedesired specificity. In a typical protocol, wells of plates (FAST,Becton-Dickinson, Palo Alto, Calif.) are coated with affinity purified,specific rabbit-anti-mouse (or suitable anti-species 1g) antibodies at10 mg/ml. The coated wells are blocked with 1% BSA, washed and exposedto supernatants from hybridomas. After incubation the wells are exposedto labeled ADEC, 1 mg/ml. Clones producing antibodies will bind aquantity of labeled ADEC which is detectable above background. Suchclones are expanded and subjected to 2 cycles of cloning at limitingdilution (1 cell/3 wells). Cloned hybridomas are injected into pristinemice to produce ascites, and monoclonal antibody is purified from mouseascitic fluid by affinity chromatography on Protein A. Monoclonalantibodies with affinities of at least 10⁸ M⁻¹ preferably 10⁹ to 10¹⁰ orstronger, will typically be made by standard procedures as described inHarlow and Lane (1988) Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory; and in Goding (1986) Monoclonal Antibodies.Principles and Practice, 2d Ed, Academic Press NYC, both incorporatedherein by reference.

X Diagnostic Test Using ADEC-Specific Antibodies

Particular ADEC antibodies are useful for the diagnosis of prepathologicconditions, and chronic or acute diseases which are characterized bydifferences in the amount or distribution of ADEC. To date, ADEC hasonly been found in inflamed adenoid and is thus specific forabnormalities or pathologies of that tissue.

Diagnostic tests for ADEC include methods utilizing the antibody and alabel to detect ADEC in human body fluids, tissues or extracts of suchtissues. The polypeptides and antibodies of the present invention may beused with or without modification. Frequently, the polypeptides andantibodies will be labeled by joining them, either covalently ornoncovalently, with a substance which provides for a detectable signal.A wide variety of labels and conjugation techniques are known and havebeen reported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent agents, chemiluminescent agents, magneticparticles and the like. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. Also, recombinant immunoglobulins may beproduced as shown in U.S. Pat. No. 4,816,567, incorporated herein byreference.

A variety of protocols for measuring soluble or membrane-bound ADEC,using either polyclonal or monoclonal antibodies specific for that ADECare known in the art. Examples include enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting(FACS). A two-site monoclonal-based immunoassay utilizing monoclonalantibodies reactive to two non-interfering epitopes on ADEC ispreferred, but a competitive binding assay may be employed. These assaysare described, among other places, in Maddox, D E et al (1983) J Exp Med158:1211.

XI Purification of Native ADEC Using Specific Antibodies

Native or recombinant ADEC was purified by immunoaffinity chromatographyusing ADEC-specific antibodies. In general, an immunoaffinity column isconstructed by covalently coupling the anti-ADEC antibody to anactivated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CNBr-activated SEPHAOSE resin (PharmaciaLKB Biotechnology, Piscataway, N.J.). The antibody is coupled to theresin, the resin is blocked, and the derivative resin is washedaccording to the manufacturer's instructions.

Such an immunoaffinity column was utilized in the purification of ADECby preparing a fraction from cells containing ADEC in a soluble form.This preparation was derived by solubilization of the whole cell or of asubcellular fraction obtained via differential centrifugation by theaddition of detergent or by other methods well known in the art.Alternatively, soluble ADEC containing a signal sequence may be secretedin useful quantity into the medium in which the cells are grown.

A soluble ADEC-containing preparation was passed over the immunoaffinitycolumn, and the column was washed under conditions that allow thepreferential absorbance of ADEC (eg, high ionic strength buffers in thepresence of detergent). Then, the column was eluted under conditionsthat disrupt antibody/ADEC binding (e.g., a buffer of pH 2-3 or a highconcentration of a chaotrope such as urea or thiocyanate ion), and theADEC was collected.

XII Determination of ADEC-Induced Chemotaxis or Cell Activation

The chemotactic activity of ADEC is measured in a 48-wellmicrochemotaxis chamber (Falk W R et al (1980) J Immunol Methods33:239). In each well, two compartments are separated by a filter thatallows the passage of cells in response to a chemical gradient. Cellculture medium such as RPMI 1640 containing ADEC is placed on one sideof a filter, usually polycarbonate, and cells suspended in the samemedia are placed on the opposite side of the filter. Sufficientincubation time is allowed for the cells to traverse the filter inresponse to the concentration gradient across the filter. Filters arerecovered from each well and cells adhering to the side of the filterfacing ADEC are typed and quantified.

The specificity of the chemoattraction is determined by performing thechemotaxis assay on specific populations of cells. First, blood cellsobtained from venipuncture are fractionated by density gradientcentrifugation and the chemotactic activity of ADEC is tested onenriched populations of neutrophils, peripheral blood mononuclear cells,monocytes and lymphocytes. Optionally, such enriched cell populationsare further fractionated using CD8+ and CD4+ specific antibodies fornegative selection of CD4+ and CD8+ enriched T-cell populations,respectively.

Another assay elucidates the chemotactic effect of ADEC on activatedT-cells. There, unfractionated T-cells or fractionated T-cell subsetsare cultured for 6 to 8 hours in tissue culture vessels coated with CD-3antibody. After this CD-3 activation, the chemotactic activity of ADECis tested as described above. Many other methods for obtaining enrichedcell populations are known in the art.

Some chemokines also produce a non-chemotactic cell activation ofneutrophils and monocytes. This is tested via standard measures ofneutrophil activation such as actin polymerization, increase inrespiratory burst activity, degranulation of the azurophilic granule andmobilization of Ca++ as part of the signal transduction pathway. Theassay for mobilization of Ca++ involves preloading neutrophils with afluorescent probe whose emission characteristics have been altered byCa++ binding. When the cells are exposed to an activating stimulus, Ca++flux is determined by observation of the cells in a fluorometer. Themeasurement of Ca++ mobilization has been described in Grynkievicz G etal. (1985) J Biol Chem 260:3440, and McColl S et al. (1993) J Immunol150:4550-4555, incorporated herein by reference.

Degranulation and respiratory burst responses are also measured inmonocytes (Zachariae C O C et al. (1990) J Exp Med 171: 2177-82).Further measures of monocyte activation are regulation of adhesionmolecule expression and cytokine production (Jiang Y et al. (1992) JImmunol 148: 2423-8). Expression of adhesion molecules also varies withlymphocyte activation (Taub D et al. (1993) Science 260: 355-358).

XIII Drug Screening

This invention is particularly useful for screening compounds by usingADEC polypeptide or binding fragment thereof in any of a variety of drugscreening techniques. The ADEC polypeptide or fragment employed in sucha test may either be free in solution, affixed to a solid support, borneon a cell surface or located intracellularly. One method of drugscreening utilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the polypeptide orfragment. Drugs are screened against such transformed cells incompetitive binding assays. Such cells, either in viable or fixed form,can be used for standard binding assays. One may measure, for example,the formation of complexes between ADEC or fragment and the agent beingtested or examine the diminution in complex formation between ADEC and aneutrophil or fibroblast caused by the agent being tested

Thus, the present invention provides methods of screening for drugs orany other agents which can affect inflammation and disease. Thesemethods comprise contacting such an agent with an ADEC polypeptide orfragment thereof and assaying (i) for the presence of a complex betweenthe agent and the ADEC polypeptide or fragment, or (ii) for the presenceof a complex between the ADEC polypeptide or fragment and the cell, bymethods well known in the art. In such competitive binding assays, theADEC polypeptide or fragment is typically labeled. After suitableincubation, free ADEC polypeptide or fragment is separated from thatpresent in bound form, and the amount of free or uncomplexed label is ameasure of the ability of the particular agent to bind to ADEC or tointerfere with the ADEC/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the ADEC polypeptidesand is described in detail in European Patent Application 84/03564,published on Sep. 13, 1984, incorporated herein by reference. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface The peptide test compounds are reacted with ADEC polypeptide andwashed. Bound ADEC polypeptide is then detected by methods well known inthe art Purified ADEC can also be coated directly onto plates for use inthe aforementioned drug screening techniques. In addition,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding ADECspecifically compete with a test compound for binding to ADECpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with ADEC.

XIV Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact, e.g., agonists, antagonists, or inhibitors. Any ofthese examples can be used to fashion drugs which are more active orstable forms of the polypeptide or which enhance or interfere with thefunction of a polypeptide in vivo (cf. Hodgson J (1991) Bio/Technology9:19-21, incorporated herein by reference).

In one approach, the three-dimensional structure of a protein ofinterest, or of a protein-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of thepolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of a polypeptide may be gained by modeling basedon the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous chemokine-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton S and Wells J A (1992 Biochemistry31:7796-7801) or which act as inhibitors, agonists, or antagonists ofnative peptides as shown by Athauda S B et al (1993 J Biochem113:742-746), incorporated herein by reference.

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anta-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amount of polypeptide maybe made available to perform such analytical studies as X-raycrystallography. In addition, knowledge of the ADEC amino acid sequenceprovided herein will provide guidance to those employing computermodeling techniques in place of or in addition to x-ray crystallography.

XV Identification of ADEC Receptors

Purified ADEC is useful for characterization and purification ofspecific cell surface receptors and other binding molecules. Cells whichrespond to ADEC by chemotaxis or other specific responses are likely toexpress a receptor for ADEC. Radioactive labels may be incorporated intoADEC by various methods known in the art. A preferred embodiment is thelabeling of primary amino groups in ADEC with ¹²⁵I Bolton-Hunter reagent(Bolton, A E and Hunter, W M (1973) Biochem J 133: 529), which has beenused to label other chemokines without concomitant loss of biologicalactivity (Hebert C A et al (1991) J Biol Chem 266: 18989; McColl S et al(1993) J Immunol 150:4550-4555). Receptor-bearing cells are incubatedwith labeled ADEC. The cells are then washed to removed unbound ADEC,and receptor-bound ADEC is quantified. The data obtained using differentconcentrations of ADEC are used to calculate values for the number andaffinity of receptors.

Labeled ADEC is useful as a reagent for purification of its specificreceptor. In one embodiment of affinity purification, ADEC is covalentlycoupled to a chromatography column. Receptor-bearing cells areextracted, and the extract is passed over the column. The receptor bindsto the column by virtue of its biological affinity for ADEC. Thereceptor is recovered from the column and subjected to N-terminalprotein sequencing. This amino acid sequence is then used to designdegenerate oligonucleotide probes for cloning the receptor gene.

In an alternate method, expression cloning, mRNA is obtained fromreceptor-bearing cells and made into a cDNA expression library. Thelibrary is transfected into a population of cells, and those cells inthe population which express the receptor are selected usingfluorescently labeled ADEC. The receptor is identified by recovering andsequencing recombinant DNA from highly labeled cells.

In another alternate method, antibodies are raised against the surfaceof receptor-bearing cells, specifically monoclonal antibodies. Themonoclonal antibodies are screened to identify those which inhibit thebinding of labeled ADEC. These monoclonal antibodies are then used inaffinity purification or expression cloning of the receptor.

Soluble receptors or other soluble binding molecules are identified in asimilar manner. Labeled ADEC is incubated with extracts or otherappropriate materials derived from inflamed adenoid. After incubation,ADEC complexes larger than the size of purified ADEC are identified by asizing technique such as size exclusion chromatography or densitygradient centrifugation and are purified by methods known in the art.The soluble receptors or binding protein(s) are subjected to N-terminalsequencing to obtain information sufficient for database identification,if the soluble protein is known, or cloning, if the soluble protein isunknown.

XVI Use and Administration of ADEC

Antibodies, inhibitors, receptors or analogs of ADEC (treatments forexcessive ADEC production, hereafter abbreviated TEC), can providedifferent effects when administered therapeutically. TECs will beformulated in a nontoxic, inert, pharmaceutically acceptable aqueouscarrier medium preferably at a pH of about 5 to 8, more preferably 6 to8, although the pH may vary according to the characteristics of theantibody, inhibitor, receptor or analog being formulated and thecondition to be treated. Characteristics of the TEC include solubilityof the molecule, half-life and antigenicity/immunogenicity and may aidin defining an effective carrier. Native human proteins are preferred asTECs, but organic molecules resulting from drug screens may be equallyeffective in particular situations.

TECs may be delivered by known routes of administration including butnot limited to topical creams or gels; transmucosal spray or aerosol,transdermal patch or bandage; injectable, intravenous or lavageformulations; or orally administered liquids or pills. The particularformulation, exact dosage, and route of administration will bedetermined by the attending physician and will vary according to eachspecific situation.

Such determinations are made by considering multiple variables such asthe condition to be treated, the TEC to be administered, and thepharmacokinetic profile of the particular TEC. Additional factors whichmay be taken into account include disease state (e.g. severity) of thepatient, age, weight, gender, diet, time of administration, drugcombination, reaction sensitivities, and tolerance/response to therapy.Long acting TEC formulations might be administered every 3 to 4 days,every week, or once every two weeks depending on half-life and clearancerate of the particular TEC.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature; see U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.It is anticipated that different formulations will be effective fordifferent TECS and that administration targeting the adenoid maynecessitate delivery in a manner different from that for deliverytargeted to a more internal tissue.

It is contemplated that conditions or diseases of the adenoids whichactivate, fibroblasts, neutrophils or other leukocytes may precipitatepermanent damage that is treatable with TECs. These conditions ordiseases may be specifically diagnosed by the tests discussed above, andsuch testing should be performed in suspected cases of Epstein-Barrvirus, Hodgkin's disease, various neoplasms or nonspecific pharyngitis.

All publications and patents mentioned in the above specification areherein incorporated by reference. The foregoing written specification isconsidered to be sufficient to enable one skilled in the art to practicethe invention. Indeed, various modifications of the above describedmodes for carrying out the invention which are obvious to those skilledin the field of molecular biology or related fields are intended to bewithin the scope of the following claims.

9 330 base pairs nucleic acid single linear cDNA Inflamed Adenoid 202931 ATGAAGTTCA TCTCGACATC TCTGCTTCTC ATGCTGCTGG TCAGCAGCCT CTCTCCAGTC 60CAAGGTGTTC TGGAGGTCTA TTACACAAGC TTGAGGTGTA GATGTGTCCA AGAGAGCTCA 120GTCTTTATCC CTAGACGCTT CATTGATCGA ATTCAAATCT TGCCCCGTGG GAATGGTTGT 180CCAAGAAAAG AAATCATAGT CTGGAAGAAG AACAAGTCAA TTGTGTGTGT GGACCCTCAA 240GCTGAATGGA TACAAAGAAT GATGGAAGTA TTGAGAAAAA GAAGTTCTTC AACTCTACCA 300GTTCCAGTGT TTAAGAGAAA GATTCCCTGA 330 109 amino acids amino acid singlelinear polypeptide Inflamed Adenoid 20293 2 Met Lys Phe Ile Ser Thr SerLeu Leu Leu Met Leu Leu Val Ser Ser 1 5 10 15 Leu Ser Pro Val Gln GlyVal Leu Glu Val Tyr Tyr Thr Ser Leu Arg 20 25 30 Cys Arg Cys Val Gln GluSer Ser Val Phe Ile Pro Arg Arg Phe Ile 35 40 45 Asp Arg Ile Gln Ile LeuPro Arg Gly Asn Gly Cys Pro Arg Lys Glu 50 55 60 Ile Ile Val Trp Lys LysAsn Lys Ser Ile Val Cys Val Asp Pro Gln 65 70 75 80 Ala Glu Trp Ile GlnArg Met Met Glu Val Leu Arg Lys Arg Ser Ser 85 90 95 Ser Thr Leu Pro ValPro Val Phe Lys Arg Lys Ile Pro 100 105 114 amino acids amino acidsingle linear peptide 3 Met Ser Leu Leu Ser Ser Arg Ala Ala Arg Val ProGly Pro Ser Ser 1 5 10 15 Ser Leu Cys Ala Leu Leu Val Leu Leu Leu LeuLeu Thr Gln Pro Gly 20 25 30 Pro Ile Ala Ser Ala Gly Pro Ala Ala Ala ValLeu Arg Glu Leu Arg 35 40 45 Cys Val Cys Leu Gln Thr Thr Gln Gly Val HisPro Lys Met Ile Ser 50 55 60 Asn Leu Gln Val Phe Ala Ile Gly Pro Gln CysSer Lys Val Glu Val 65 70 75 80 Val Ala Ser Leu Lys Asn Gly Lys Glu IleCys Leu Asp Pro Glu Ala 85 90 95 Pro Phe Leu Lys Lys Val Ile Gln Lys IleLeu Asp Gly Gly Asn Lys 100 105 110 Glu Asn 107 amino acids amino acidsingle linear peptide 4 Met Ala Arg Ala Thr Leu Ser Ala Ala Pro Ser AsnPro Arg Leu Leu 1 5 10 15 Arg Val Ala Leu Leu Leu Leu Leu Leu Val AlaAla Ser Arg Arg Ala 20 25 30 Ala Gly Ala Pro Leu Ala Thr Glu Leu Arg CysGln Cys Leu Gln Thr 35 40 45 Leu Gln Gly Ile His Leu Lys Asn Ile Gln SerVal Lys Val Lys Ser 50 55 60 Pro Gly Pro His Cys Ala Gln Thr Glu Val IleAla Thr Leu Lys Asn 65 70 75 80 Gly Gln Lys Ala Cys Leu Asn Pro Ala SerPro Met Val Lys Lys Ile 85 90 95 Ile Glu Lys Met Leu Lys Asn Gly Lys SerAsn 100 105 106 amino acids amino acid single linear peptide 5 Met AlaHis Ala Thr Leu Ser Ala Ala Pro Ser Asn Pro Arg Leu Leu 1 5 10 15 ArgVal Ala Leu Leu Leu Leu Leu Leu Val Gly Ser Arg Arg Ala Ala 20 25 30 GlyAla Ser Val Val Thr Glu Leu Arg Cys Gln Cys Leu Gln Thr Leu 35 40 45 GlnGly Ile His Leu Lys Asn Ile Gln Ser Val Asn Val Arg Ser Pro 50 55 60 GlyPro His Cys Ala Gln Thr Glu Val Ile Ala Thr Leu Lys Asn Gly 65 70 75 80Lys Lys Ala Cys Leu Asn Pro Ala Ser Pro Met Val Gln Lys Ile Ile 85 90 95Glu Lys Ile Leu Asn Lys Gly Ser Thr Asn 100 105 99 amino acids aminoacid single linear peptide 6 Met Thr Ser Lys Leu Ala Val Ala Leu Leu AlaAla Phe Leu Ile Ser 1 5 10 15 Ala Ala Leu Cys Glu Gly Ala Val Leu ProArg Ser Ala Lys Glu Leu 20 25 30 Arg Cys Gln Cys Ile Lys Thr Tyr Ser LysPro Phe His Pro Lys Phe 35 40 45 Ile Lys Glu Leu Arg Val Ile Glu Ser GlyPro His Cys Ala Asn Thr 50 55 60 Glu Ile Ile Val Lys Leu Ser Asp Gly ArgGlu Leu Cys Leu Asp Pro 65 70 75 80 Lys Glu Asn Trp Val Gln Arg Val ValGlu Lys Phe Leu Lys Arg Ala 85 90 95 Glu Asn Ser 107 amino acids aminoacid single linear peptide 7 Met Ala Arg Ala Ala Leu Ser Ala Ala Pro SerAsn Pro Arg Leu Leu 1 5 10 15 Arg Val Ala Leu Leu Leu Leu Leu Leu ValAla Ala Gly Arg Arg Ala 20 25 30 Ala Gly Ala Ser Val Ala Thr Glu Leu ArgCys Gln Cys Leu Gln Thr 35 40 45 Leu Gln Gly Ile His Pro Lys Asn Ile GlnSer Val Asn Val Lys Ser 50 55 60 Pro Gly Pro His Cys Ala Gln Thr Glu ValIle Ala Thr Leu Lys Asn 65 70 75 80 Gly Arg Lys Ala Cys Leu Asn Pro AlaSer Pro Ile Val Lys Lys Ile 85 90 95 Ile Glu Lys Met Leu Asn Ser Asp LysSer Asn 100 105 101 amino acids amino acid single linear peptide 8 MetSer Ser Ala Ala Gly Phe Cys Ala Ser Arg Pro Gly Leu Leu Phe 1 5 10 15Leu Gly Leu Leu Leu Leu Pro Leu Val Val Ala Phe Ala Ser Ala Glu 20 25 30Ala Glu Glu Asp Gly Asp Leu Gln Cys Leu Cys Val Lys Thr Thr Ser 35 40 45Gln Val Arg Pro Arg His Ile Thr Ser Leu Glu Val Ile Lys Ala Gly 50 55 60Pro His Cys Pro Thr Ala Gln Leu Ile Ala Thr Leu Lys Asn Gly Arg 65 70 7580 Lys Ile Cys Leu Asp Leu Gln Ala Pro Leu Tyr Lys Lys Ile Ile Lys 85 9095 Lys Leu Leu Glu Ser 100 109 amino acids amino acid single linearpeptide 9 Met Lys Phe Ile Ser Thr Ser Leu Leu Leu Met Leu Leu Val SerSer 1 5 10 15 Leu Ser Pro Val Gln Gly Val Leu Glu Val Tyr Tyr Thr SerLeu Arg 20 25 30 Cys Arg Cys Val Gln Glu Ser Ser Val Phe Ile Pro Arg ArgPhe Ile 35 40 45 Asp Arg Ile Gln Ile Leu Pro Arg Gly Asn Gly Cys Pro ArgLys Glu 50 55 60 Ile Ile Val Trp Lys Lys Asn Lys Ser Ile Val Cys Val AspPro Gln 65 70 75 80 Ala Glu Trp Ile Gln Arg Met Met Glu Val Leu Arg LysArg Ser Ser 85 90 95 Ser Thr Leu Pro Val Pro Val Phe Lys Arg Lys Ile Pro100 105

What is claimed is:
 1. An isolated antibody selected from the groupconsisting of: a) an antibody which specifically binds to a polypeptidecomprising the amino acid sequence of SEQ ED NO:2, wherein the antibodybinds to an epitope of a polypeptide consisting of the amino acidsequence of SEQ ID NO:2, b) an antibody which specifically binds to apolypeptide comprising a naturally occurring ammo acid sequence at least90% identical to the amino acid sequence of SEQ ID NO:2, wherein thepolypeptide has chemotactic activity or is able to activate neutrophilsor monocytes, and wherein the antibody binds to an epitope of apolypeptide consisting of a naturally occurring amino acid sequence atleast 90% identical to SEQ ID NO:2, and c) an antibody whichspecifically binds to a fragment comprising at least 9 contiguous aminoacids of a polypeptide consisting of the amino acid sequence of SEQ IDNO:2, wherein the fragment has chemotactic activity or is able toactivate neutrophils or monocytes.
 2. The antibody of claim 1, whereinthe antibody is: a) a chimeric antibody, b) a single chain antibody, c)a Fab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 3. Acomposition comprising an antibody of claim 1 and an acceptableexcipient.
 4. A composition of claim 3, wherein the antibody is labeled.5. A method of preparing an antibody of claim 1, wherein the antibody isa polyclonal antibody, the method comprising: a) immuring an animal witha polypeptide having the ammo acid sequence of SEQ ID NO:2, or animmunogenic fragment comprising at least 9 contiguous amino acids of SEQID NO:2, under conditions to elicit an antibody response, b) isolatingantibodies from said animal, and c) screening the isolated antibodieswith the polypeptide, thereby identifying a polyclonal antibody whichbinds specifically to a polypeptide having the amino acid sequence ofSEQ ID NO:2.
 6. An antibody produced by a method of claim
 5. 7. Acomposition comprising the antibody of claim 6 and a suitable carrier.8. A method of making an antibody of claim 1, wherein the antibody is amonoclonal antibody, the method comprising: a) immunizing an animal witha polypeptide having the amino acid sequence of SEQ ID NO:2, or animmunogenic fragment comprising at least 9 contiguous amino acids of SEQID NO2, under conditions to elicit an antibody response, b) isolatingantibody producing cells from the animal, c) fusing the antibodyproducing cells with immortalized cells to form monoclonalantibody-producing hybridoma cells, d) culturing the hybridoma cells,and e) isolating from the culture monoclonal antibody which bindsspecifically to a polypeptide having the amino acid sequence of SEQ IDNO:2.
 9. A monoclonal antibody produced by a method of claim
 8. 10. Acomposition comprising the monoclonal antibody of claim 9 and a suitablecarrier.
 11. The antibody of claim 1, wherein the antibody is producedby screening a Fab expression library.
 12. The antibody of claim 1,wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 13. A method of detecting a polypeptide havingthe amino acid sequence of SEQ ID NO:2 in a sample, the methodcomprising: a) incubating the antibody of claim 1 with a sample underconditions to allow specific binding of the antibody and thepolypeptide, and b) detecting specific binding, wherein specific bindingindicates the presence of a polypeptide having the amino acid sequenceof SEQ ID NO:2 in the sample.
 14. A method of purifying a polypeptidehaving the amino acid sequence of SEQ ID NO:2 from a sample, the methodcomprising: a) incubating the antibody of claim 1 with a sample underconditions to allow specific binding of the antibody and thepolypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide having the amino acid sequence of SEQID NO:2.